Synthetic Protein Research Articles

Synthetic Protein Scaffolds for Improving R-(-)-Linalool Production in Escherichia coli.

R-(-)-Linalool is widely used in the pharmaceutical, agrochemical, and fragrance industries; however, its applications are limited owing to low yield and high cost of production. To improve the production efficiency of R-(-)-linalool in Escherichia coli, three enzymes [E. coli-derived isopentenyl diphosphate isomerase, Abies grandis-derived geranyl diphosphate synthase, and Streptomyces clavuligerus-derived (3R)-linalool synthases] were physically colocalized to synthetic complexes using synthetic protein scaffolds of GTPase-binding domain, Src homology 3, and PSD95/DlgA/Zo-1. R-(-)-Linalool was produced at the highest concentration in the strain IGL114 containing a scaffold ratio of 1:1:4. By further optimizing the inducer, temperature, and glycerol concentration, the production titer of R-(-)-linalool in the shake flask was increased by approximately 10 times compared with that of the scaffold-free control and was 2.78 times the previously reported yield. The production in the fermenter was about 1.5 times the previous highest production. In general, the final strain accumulated 277.8 and 1523.2 mg/L R-(-)-linalool under the conditions of shake-flask and fed-batch fermentation, respectively. This study provides a foundation for the assembly of bacterial intracellular protein scaffolds.

Self-driving armored CAR-T cells overcome a suppressive milieu and eradicate CD19+ Raji lymphoma in preclinical models

Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion <i>in vitro</i> as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor β receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-β on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.

Influence of Hydroxyproline on Mechanical Behavior of Collagen Mimetic Proteins Under Fraying Deformation-Molecular Dynamics Investigations.

Molecular dynamics modeling is used to simulate, model, and analyze mechanical deformation behavior and predictive properties of three different synthetic collagen proteins obtained from RSC-PDB, 1BKV, 3A08, and 2CUO, with varying concentrations of hydroxyproline (HYP). Hydroxyproline is credited with providing structural support for the collagen protein molecules. Hydroxyproline's influence on these three synthetic collagen proteins' mechanical deformation behavior and predictive properties is investigated in this paper. A detailed study and inference of the protein's mechanical characteristics associated with HYP content are investigated through fraying deformation behavior. A calculated Gibbs free energy value (ΔG) of each polypeptide α chain that corresponds with a complete unfolding of a single polypeptide α-chain from a triple-helical protein is obtained with umbrella sampling. The force needed for complete separation of the polypeptide α-chain from the triple-helical protein is analyzed for proteins to understand the influence of HYP concentration and is discussed in this paper. Along with a difference in ΔG, different unfolding pathways for the molecule and individual chains are observed. The correlation between the fraying deformation mechanical characteristics and the collagen proteins' hydroxyproline content is provided in this study via the three collagen proteins' resulting binding energies.

PPARɣ Agonist Pioglitazone and Synthetic Surfactant Protein B Mimic B‐YL Prevents Hyperoxia‐Induced Lung Injury in Adult Mice

Purpose of Study Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS) are serious conditions and often life threatening. ALI and ARDS are characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction. Previously we demonstrated that perturbation in TGF-β and wingless/Int (Wnt) signaling pathways, known to be involved in perinatal lung development and hyperoxia-induced rat lung injury, was ameliorated by pioglitazone and B-YL, a novel synthetic surfactant, in neonatal rats. However, whether this approach can be extended to be effective in adult lung injury is unknown. We hypothesize that by stabilizing alveolar homeostasis, combined PPARγ agonist PGZ and oxidant resistant synthetic surfactant B-YL can be a unified approach to block hyperoxia-induced lung injury not only in neonates but also in adults. Methods Used 3 to 6 months old C57BL/6 adult mice lungs were harvested. Lung explants were placed in 15% FBS/Weymouth medium and exposed ex-vivo to 21% or 95% O2 for 24 or 72h. Treatment groups included: 1) untreated control; 2) B-YL (100 mg/kg); 3) PGZ (1 mg/kg); and 4) B-YL (100 mg/kg)+PGZ (1 mg/kg). Wnt signaling (LEF-1 and β-catenin), TGF-β signaling (Alk5 and SMAD3), and apoptosis (BAX) markers were determined by Western analysis. Inflammatory cytokines (IL6, IL1β, TNFα, and MCP1) were measured by qRT-PCR. Summary of Results Hyperoxia exposure resulted in significant increases in lung injury and apoptosis markers including the upregulation of Wnt and TGF-β pathway proteins. PGZ+ B-YL treated group showed significant amelioration of hyperoxia-induced alterations in these injury markers, including Wnt (LEF-1 and β-catenin) and TGF-β (ALK-5) pathway proteins (vs. 95% O2 control group). Similarly, hyperoxia-induced increases in IL6, IL1β, TNFα, and MCP1 were blocked in B-YL alone, as well as PGZ+B-YL treated groups. Conclusions Effectiveness of combined PGZ+B-YL in blocking hyperoxia- induced lung injury ex-vivo sets the stage to test this therapeutic approach for adult lung injury in vivo.

A Sos proteomimetic as a pan-Ras inhibitor

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.

O16: TARGETING THE LOX-1 SCAVENGER RECEPTOR ATTENUATES ATHEROSCLEROSIS AND NEOINTIMAL HYPERPLASIA IN APO-E NULL TRANSGENIC MICE

Abstract Introduction The clinical consequences of atherosclerosis and post-procedure neointimal hyperplasia (NIH) represent a significant proportion of the vascular surgeon's workload. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a class E scavenger receptor expressed in endothelial cells implicated in atherosclerosis and NIH. This study aims to demonstrate the therapeutic potential of LOX-1 targeting using a transgenic mouse model and LOX-1-specific non-antibody synthetic protein scaffolds called affimers. Method Apolipoprotein-E (APO-E) null and APO-E/LOX-1 double-null transgenic mice were fed a high cholesterol western diet for 12 weeks. One group of APO-E null mice was treated subcutaneously with LOX-1 affimer. All mice underwent unilateral common femoral artery wire injury at week 8. Mice were culled and tissues harvested for evaluation of atherosclerotic burden and femoral artery NIH. Studies were carried out with Home Office approval. Result Compared with APO-E null controls, carotid and femoral bifurcation atherosclerosis (p&amp;lt;0.05) and total aortic plaque coverage (p&amp;lt;0.01) were significantly reduced in APO-E/LOX-1 double-null mice. Reductions were observed in APO-E null mice treated with LOX-1 affimer, however these were non-significant. Femoral artery NIH was reduced by 16% in APO-E/LOX-1 double null and 11% in LOX-1 affimer treated APO-E null mice compared with untreated APO-E controls, however these reductions were non-significant. Conclusion This study demonstrates the therapeutic potential of LOX-1 targeting in large vessel atherosclerotic disease and NIH. Knockout of the LOX-1 transgene produced promising results, while results following LOX-1 affimer therapy were less impressive. Improvements in affimer pharmacokinetics and drug delivery optimisation are avenues for future studies. Take-home message The LOX-1 scavenger receptor is a potential novel therapeutic target in large-vessel atherosclerosis and neointimal hyperplasia.

Synthetic Protein Condensates That Inducibly Recruit and Release Protein Activity in Living Cells.

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA-Based Recruitment Domains.

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA‐Based Recruitment Domains

AbstractHexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Study the lipidoid nanoparticle mediated genome editing protein delivery using 3D intestinal tissue model

Lipid nanoparticles are promising carriers for oral drug delivery. For bioactive cargos with intracellular targets, e.g. gene-editing proteins, it is essential for the cargo and carrier to remain complexed after crossing the epithelial layer of intestine in order for the delivery system to transport the cargos inside targeted cells. However, limited studies have been conducted to verify the integrity of cargo/carrier nanocomplexes and their capability in facilitating cargo delivery intracellularly after the nanocomplex crossing the epithelial barrier. Herein, we used a traditional 2D transwell system and a recently developed 3D tissue engineered intestine model and demonstrated the synthetic lipid nanoparticle (carrier) and protein (cargo) nanocomplexes are able to cross the epithelial layer and deliver the protein cargo inside the underneath cells. We found that the EC16-63 LNP efficiently encapsulated the GFP-Cre recombinase, penetrated the intestinal monolayer cells in both the 2D cell culture and 3D tissue models through temporarily interrupting the tight junctions between epithelial layer. After transporting across the intestinal epithelia, the EC16-63 and GFP-Cre recombinase nanocomplexes can enter the underneath cells to induce gene recombination. These results suggest that the in vitro 3D intestinal tissue model is useful for identifying effective lipid nanoparticles for potential oral drug delivery.

Development of a chimeric vaccine candidate based on Toxoplasma gondii major surface antigen 1 and apicoplast proteins using comprehensive immunoinformatics approaches

This study was aimed at designing and evaluation of a multimeric vaccine construct against Toxoplasma gondii via utilization of SAG1 along with apicoplast ribosomal proteins (S2, S5 and L11). Top-ranked MHC-I and MHC-II binding as well as shared, immunodominant linear B-cell epitopes were predicted and joined together via appropriate linkers. Also, TLR-4 agonist (RS-09 synthetic protein) and His-tag were added to the N- and C-terminal of the vaccine sequence. The finally-engineered chimeric vaccine had a length of 291 amino acids with a molecular weight of 31.46 kDa. Physico-chemical features showed a soluble, highly-antigenic and non-allergenic candidate. Secondary and tertiary structures were predicted, and subsequent analyses confirmed the construct stability that was capable to properly interact with human TLR-4. Immunoinformatics-based simulation displayed potent stimulation of T- and B-cell mediated immune responses upon vaccination with the proposed multi-epitope candidate. In conclusion, obtained information demonstrated a highly antigenic vaccine candidate, which could develop high levels of IFN-γ and other components of cellular immune profile, and can be directed for toxoplasmosis prophylactic purposes.

Strategy for the Enrichment of Protein Biomarkers from Diverse Bacterial Select Agents.

Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation. A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario. Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein. The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested. The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.

Tryptanthrin Regulates Vascular Smooth Muscle Cell Phenotypic Switching in Atherosclerosis by AMP-Activated Protein Kinase/Acetyl-CoA Carboxylase Signaling Pathway.

Atherosclerosis (AS) is one of the most severe cardiovascular diseases involved in the phenotypic switching of vascular smooth muscle cells (VSMCs). Tryptanthrin is a natural product with broad biological activities. However, the effect of tryptanthrin on atherosclerotic progression is unclear. The aim of this study was to determine the role of tryptanthrin in AS and explore the potential mechanism. In vitro, primary VSMCs were stimulated with platelet-derived growth factor-BB (PDGF) to induce cell dedifferentiation. Treatment with tryptanthrin (5 μM or 10 μM) suppressed the proliferation and recovered the contractility of VSMCs in the presence of PDGF. The contractile proteins (α-smooth muscle actin, calponin, and SM22α) were increased, and the synthetic protein vimentin was decreased by tryptanthrin in PDGF-induced VSMCs. ApoE-/- mice fed with high-fat diet were used as an in vivo model of AS. Similarly, gavage administration of tryptanthrin (50 mg/kg or 100 mg/kg) attenuated VSMC phenotypic changes from a contractile to a synthetic state in aortic tissues of AS mice. The serum lipid level, atherosclerotic plaque formation, and arterial intimal hyperplasia were attenuated by tryptanthrin. Furthermore, tryptanthrin increased the expression levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) both in vitro and in vivo. Administration of compound C, an AMPK inhibitor, reversed the inhibitory effect of tryptanthrin on VSMC dedifferentiation in vitro. Thus, we demonstrate that tryptanthrin protects against AS progression through the inhibition of VSMC switching from a contractile to a pathological synthetic phenotype by the activation of AMPK/ACC pathway. It provides novel insights into AS prevention and treatment.

Organizing Multi-Enzyme Systems into Programmable Materials for Biocatalysis

Significant advances in enzyme discovery, protein and reaction engineering have transformed biocatalysis into a viable technology for the industrial scale manufacturing of chemicals. Multi-enzyme catalysis has emerged as a new frontier for the synthesis of complex chemicals. However, the in vitro operation of multiple enzymes simultaneously in one vessel poses challenges that require new strategies for increasing the operational performance of enzymatic cascade reactions. Chief among those strategies is enzyme co-immobilization. This review will explore how advances in synthetic biology and protein engineering have led to bioinspired co-localization strategies for the scaffolding and compartmentalization of enzymes. Emphasis will be placed on genetically encoded co-localization mechanisms as platforms for future autonomously self-organizing biocatalytic systems. Such genetically programmable systems could be produced by cell factories or emerging cell-free systems. Challenges and opportunities towards self-assembling, multifunctional biocatalytic materials will be discussed.

Large scale production of synthetic spider silk proteins in Escherichia coli

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.

Impact of Arginine–Phosphate Interactions on the Reentrant Condensation of Disordered Proteins

Re-entrant condensation results in the formation of a condensed protein regime between two critical ion concentrations. The process is driven by neutralization and inversion of the protein charge by oppositely charged ions. Re-entrant condensation of cationic proteins by the polyvalent anions, pyrophosphate and tripolyphosphate, has previously been observed, but not for citrate, which has similar charge and size compared to the polyphosphates. Therefore, besides electrostatic interactions, other specific interactions between the polyphosphate ions and proteins must contribute. Here, we show that additional attractive interactions between arginine and tripolyphosphate determine the re-entrant condensation and decondensation boundaries of the cationic, intrinsically disordered saliva protein, histatin 5. Furthermore, we show by small-angle X-ray scattering (SAXS) that polyvalent anions cause compaction of histatin 5, as would be expected based solely on electrostatic interactions. Hence, we conclude that arginine–phosphate-specific interactions not only regulate solution properties but also influence the conformational ensemble of histatin 5, which is shown to vary with the number of arginine residues. Together, the results presented here provide further insight into an organizational mechanism that can be used to tune protein interactions in solution of both naturally occurring and synthetic proteins.

Predictive Platforms of Bond Cleavage and Drug Release Kinetics for Macromolecule–Drug Conjugates

Macromolecule-drug conjugates (MDCs) occupy a critical niche in modern pharmaceuticals that deals with the assembly and combination of a macromolecular carrier, a drug cargo, and a linker toward the creation of effective therapeutics. Macromolecular carriers such as synthetic biocompatible polymers and proteins are often exploited for their inherent ability to improve drug circulation, prevent off-target drug cytotoxicity, and widen the therapeutic index of drugs. One of the most significant challenges in MDC design involves tuning their drug release kinetics to achieve high spatiotemporal precision. This level of control requires a thorough qualitative and quantitative understanding of the bond cleavage event. In this review, we highlight specific research findings that emphasize the importance of establishing a precise structure-function relationship for MDCs that can be used to predict their bond cleavage and drug release kinetic parameters.